Table 1.

Methods of fabrication of tissue engineering systems, particularly applied for NTE.

Techniques	Structural characteristics	Advantages	Disadvantages	Potential impact in NTE
Conventional methods
Solvent casting/particulate leaching [9]	Through the control of the amount of porogen added, as well as its size and shape, these scaffolds usually have an average pore size of ∼500 ​μm with ∼95% porosity.	-Simple, easy, inexpensive -Does not require large equipment -Controllable pore size -Less amount of polymer required for scaffold creation	-Scaffolds may retain some toxicity -Time-consuming -Pore shape and interpore openings cannot be controlled. -Can only be used to produce thin membranes (≤3-mm thickness)	Fabrication of biocompatible scaffolds for peripheral nerve injury repair, in combination with molding techniques
Phase separation (non-solvent–induced phase separation [NIPS] [10] and thermally induced phase separation [TIPS] [11])	Porous scaffolds with possible integration of bioactive molecules	-Can produce porous scaffolds with integrated bioactive molecules owing to the low temperatures used during fabrication -No decrease in the activity of the molecule -Can be easily combined with other fabrication methods (i.e., particulate leaching, rapid prototyping)	-Difficult to control the precise scaffold morphology -Limited material selection	Fabrication of scaffolds, in conjunction with molding techniques
Self-assembly [12]	Nanofibers with amino acid residues that can be modified by the addition of bioactive molecules	-Production of very thin nanofibers -The nanofibers have amino acid residues that can be chemically modified by the addition of bioactive moieties. -Does not require organic solvents -Reduction of cytotoxicity due to the use of aqueous salt solutions or physiological media	-Complicated and elaborate process -Poor mechanical stability makes it difficult to create stable 3D structures. -The engineered nanofibers can be fragmented and are susceptible to endocytosis. -High cost of synthesis	Formation of injectable materials for nerve regeneration
Freeze-drying [13]	Porous scaffolds without the presence of potentially harmful solvents	-Controllable pore size through controlling the freezing rate and pH -Does not require high temperatures -Does not require a separate leaching step	-Small pore sizes -Long processing times -Difficult to produce scaffolds with hierarchical structures (e.g., vascularized systems)	Design of scaffolds for nerve repair using biocompatible and biodegradable materials
Gas foaming [14]	Creation of porous scaffolds with pore sizes ranging from 100 to 500 ​μm	-Does not require the use of organic solvents and high temperatures -Controllable porosity dependent on the amount of gas dissolved in the polymer	-Limited mechanical property control -Possibility of creating a non-porous external surface	Fabrication of scaffolds, in conjunction with other techniques (i.e., molding, phase separation)
Hydrogel formation [15]	Hydrogels can have a range of different properties that depend on the type of polymeric material used and the method of cross-linking used.	-Tunable mechanical strength -Mostly biocompatible and biodegradable -Can offer controllable drug release rates -Incorporation of biological material (DNA, proteins, cells)	-Potential lack of biodegradability for some materials -Difficulty with drug loading in cases of non-hydrophilic drugs -Potential toxicity from unreacted cross-linking molecules	Fabrication of scaffolds using bioprinting methods and design of bioinks for brain delivery and regeneration
Molding and texturing methods
Compression molding/injection molding [9]	Scaffolds with controllable porosity through the use of porogens with different sizes and chemical properties	-Control of pore size, interconnectivity, and geometry -Does not require organic solvents	-High temperatures required for non-amorphous polymers -Possibility of residual porogens	Construction of in vitro brain models for drug screening and efficacy testing (such as lab-on-a-chip devices). Should be used in combination with other methods
Photolithography [16]	Scaffolds with details in the nanometer and micrometer scale printed on photoresists	-Large-scale patterning -High-resolution technique -Non-contact manufacturing process	-High cost -Limited control over surface properties -Compatible with a limited number of materials	–
Soft lithography [17]	Scaffolds with details in the nanometer and micrometer scale that have been transferred onto a range of polymers with different properties	-Wide range of materials -Easy and straightforward process -Low cost	-Can lose some of the detail during the stamp/mold creation process	Fabrication of components for microfluidic systems and lab-on-a-chip devices. Like compression molding, should be used in combination with other methods.
Laser texturing [18]	Structuring/texturing of the material surface can be localized without affecting the surrounding areas and is in the region of nanometers and micrometers	-Local excitation of certain areas of a material, with minimal damage to the surrounding areas -Non-contact fabrication method -Can be used with a wide range of materials	-Requires specialized and expensive equipment	Precise and controllable micropatterning/nanopatterning of scaffolds for nerve regeneration
Fiber mesh/fiber bonding [19]	Fibrous scaffolds with large surface areas	-Creation of scaffolds with large surface areas -The mesh structure allows rapid diffusion of nutrients. -Mechanical stability	-Lacks structural stability -Poor mechanical property control -Limited applications in other polymers (except for PGA, PLLA)	–
Electrohydrodynamic techniques
Electrospraying [20]	Highly charged droplets are formed. Their charge prevents their coagulation and promotes their self-dispersion.	-The size distribution of the droplets is usually narrow; no droplet agglomeration and coagulation are occurring. -The motion of charged droplets can be easily controlled. -Higher deposition efficiency of charged spray than that of uncharged droplets -Single-step processing	-Requires specialized equipment -Difficult to control the droplet's size -Can induce macromolecule degradation	Fabrication of carriers for drugs and therapeutic molecules for brain delivery.
Electrospinning [21]	Continuous microscale and nanoscale fibers from a rich variety of materials. By blending different polymers, nanofibers with internal morphology, and secondary structures, e.g., porous, hollow, or core-sheath structure can be fabricated. In addition, fibers can be organized into ordered arrays or hierarchical structures by modulating their stacking, arrangement, and folding.	-Easy to use, simple, versatile, efficient, and ideal for large-scale production -High surface area-to-volume ratio structures -Large number of interfibrous/intrafibrous pores (high porosity) -Fabrication of fibers from various types of raw materials (from natural and synthetic polymers to composites, consisting of organic and inorganic components) leads to unlimited applications -The ability to control many factors, such as the fiber diameter, orientation, and composition	-The requirement for specialized equipment (although it is inexpensive) -The use of organic solvents -The limited control of pore structures -The process depends on many variables	Synthetic nerve conduits to facilitate axonal guidance and to enhance nerve regeneration
Solid freeform fabrication/rapid prototyping
Photolithography-based techniques [22]	Precise internal architectures and external geometries, which match those of human tissue (structures with ≥50-μm features)	-Good mechanical strength -High spatial resolution -Easy to achieve small features -High degree of fabrication accuracy -Low printing time -Bioresins can be incorporated to create bioinks.	-Resins with cytotoxic residuals may be used. -Often during the processing, supportingstructures are required. -Specialized equipment is required. -High-intensity UV light may be used.	Fabrication of complex 3D tissue structure with high resolution for brain regeneration as long as biocompatible hydrogels are used
Selective laser sintering/selective laser melting [23]	Fabrication of complex geometries with intricate and controllable internal architectures	-Good mechanical strength -High accuracy -Broad range of materials can be used. -Easy to create layered 3D structures ​as new layers of powder can be layered on top of the previous sintered layers. -No supports required -Many commercial machine providers exist.	-Elevated temperatures -Local high energy input -Uncontrolled porosity -Laser beam diameter (∼400 ​μm) and powder particle size limit the dimension of the generated scaffolds. -Difficulty in building specially shaped scaffolds with sharp corners or clear boundaries -Restricted by material properties; (The material has to be available as a powder, and the powder must have suitable melting and welding behaviour.)	–
Microsphere sintering (subcategory of sintering) [24]	Microspheres are fused together to create a single macroscopic unit, with complex shapes and architectures.	-Can load biological material into the water droplets -Suitable for obtaining composite structures from polymeric and inorganic substances -Customization (patient-specific) of the scaffolds -Freedom from toxic solvents	-Very expensive ​because large quantities of raw materials are needed -Requires specialized equipment -Microspheres may stick together or may not spread evenly in successive layers	Fabrication of macroporous, 3D shape–specific constructs, conductive to infiltration and with controlled release of bioactive molecules for nerve regeneration
Fused deposition modeling [25]	Scaffolds with honeycomb-like pattern, fully interconnected channel network, and controllable porosity and channel size	-Low cost -Good mechanical strength -Versatile pattern design -Does not require any solvent	-Elevated temperatures -Small range of bulk materials -Thermal degradation and spatial resolution	New biocompatible and biodegradable filament materials must be formulated to use fused deposition modeling for nerve regeneration applications.
3D bioprinting [26,27]	Precise layering of cells, biologic scaffolds, and growth factors to create bioidentical tissue for a variety of uses.	-Broad range of materials and conditions -Incorporation of cells and macromolecules -Accurate reproduction of tissue -Potential of an industry-scale robotic tissue-fabrication line	-Slow processing -Time-consuming -High cost and size -Low mechanical strength -Low process resolution -Lack of full automation -Cartridges and nozzles, whenever they are used, can negatively affect cell viability.	Construction of brain-like structures to serve as in vitro 3D models and custom-made platforms for personalized medicine

NTE, neural tissue engineering; PGA, polyglycolic acid; PLLA, poly-l-lactic acid; 3D, three-dimensional.